The ordered packaging of DNA in chromosomes and the organization of chromosomes in the nucleus is important for proper regulation during growth, development and differentiation of organisms. Chromosomes in the nucleus occupy specific territories, which are further subdivided into transcriptionally active and inactive domains. Gene regulatory elements, such as enhancers and silencers, function to activate and inactivate genes and these elements function within the context of these domains. In this grant proposal we wish to identify the DNA sequence elements as well as the proteins that regulate the proper packaging and organization of chromosomes. DNA elements called insulators are necessary for the partitioning of chromosomes into domains and these elements also function to organize the domains within the three-dimensional context of the nucleus. Understanding how DNA elements and chromatin domains interact physically and functionally with other elements and domains is the second focus of this proposal. We wish to determine the role of tRNA genes in organizing chromosome structure. We wish to determine the mechanisms by which tRNA genes function in chromatin organization in human cells and lastly we wish to determine how DNA repair proteins help organize inactive chromatin domains in the nucleus. The research will involve use of molecular genetics and genomics, manipulation of chromatin using techniques like quantitative chromatin immunoprecipitations, and chromosome conformation capture. Understanding how the nucleus is organized should help us better manipulate processes for benefit in research and medicine. The results from this study will be useful in understanding the molecular basis of disease states that arise from mis-regulation of chromatin domains. The results will also enable researchers to develop new strategies for treating disease states by developing new molecular tools for gene therapy.